The long-term objective of this research project is to analyze the cellular and synaptic mechanisms of an example of associative learning and memory produced by Pavlovian conditioning. The studies proposed in this grant renewal will provide insights into general principles underlying memory following one-trial and multi-trial conditioning, and contribute to the elucidation of events that are essential for the transformation of memory into an enduring form;long-term memory. To address these goals, a multi-disciplinary approach to the study of associative learning and memory will be carried out in the marine mollusk Hermissenda, a preparation that has proven useful in biophysical, biochemical, and molecular studies of associative learning. The research will use a combination of neurophysiological, biophysical, biochemical, molecular, proteomics, and behavioral techniques to study short- and long-term memory in intact animals, isolated identified sensory neurons and interneurons of the CS pathway, and isolated nervous systems. One primary goal of the research is to identify proteins whose synthesis and/or phosphorylation are regulated by one-trial and multi-trial Pavlovian conditioning. During the previous period of support we identified and characterized Csp24, a protein consisting of beta-thymosin repeats whose phosphorylation is regulated by one-trial and multi-trial Pavlovian conditioning. The widespread beta-thymosin actin binding domain has regulatory properties in actin dynamics and recently has been shown to contribute in morphogenesis and memory. We have recently cloned the full-length Csp24 cDNA and showed that blocking Csp24 expression inhibits the development of intermediate-term memory. Studies are proposed to determine the specific role of phosphorylated Csp24 in time dependent processes of memory consolidation using site-directed mutagenesis of the identified phosphorylation sites of Csp24. We will determine if conditioning modifies the distribution of Csp24 in the soma, axonal, or synaptic compartments of identified sensory neurons, and examine the regulation of Csp24 by several signal transduction pathways that are associated with conditioning. We will examine the two specific examples of plasticity in identified cells of conditioned animals;synaptic facilitation and intrinsic enhanced excitability. A goal of this project is to identify basic mechanisms for extinction of Pavlovian conditioning and the expression of savings following re-acquisition. Collectively, these studies of learning and memory will help to elucidate basic mechanisms of short- and long-term memory and provide insights into the relationship between memory mechanisms and clinical disorders of memory .